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PHYSIOLOGIA PLANTARUM 100: 716-728. 1997 Copyrigh,eph,siobgu,pimianm m?

Primed in Denmark - all rights resen-ed JSSN 0031-9317

Cellular 'elementary organisms' in vitro. The early vision of


Gottlieb Haberlandt and its realization
Ekkehard Hoxtermann

Hoxtermann, E. 1997. Cellular 'elementarj' organisms' in vitro. The early vision of


Gottlieb Haberlandt and its realization. - Physiol. Plant. iOO: 716-728.

The botanist Gottlieb Haberlandt was. the first who in 1898 (published in 1902) tried
the systematic culture in vitro of single cells. His purpose was to study the mutual in-
fluences of cells as the last 'living units' within the multicellular body. Haberlandt vi-
saalized the theoretical potential of the culture approach in experimental plant mor-
phology and physiolQ.gy and nearly half a century was to elapse before his far-reach-
ing ideas were reahzed. The history of cell and tissue culture is traced from the first
concepts and origins in experimental embryology (1858/59) lo its final verification al-
most 100 years later. The paper reminds of essential steps, theoretical backgrounds,
accidental and planned discoveries and methodological approaches, and attempts to
present all sources. The more or less known reviews of White (1931, 1936 and 1946),
Fiedler (1938/39), Stapp (1947), Kandler (1948) and Street (1959) are summarized,
and the mutual influence of plant and animal tissue culture on each other is discussed.
Previous retrospective articles on the particular role of Haberlandt, namely those of
Gaulheret (1942) and Krikorian and Berquam (1969), are included in a general assess-
ment of physiological anatomy in memory of the 50th anniversary of this great bota-
nist's death.

Key words - Cells as 'elementary organisms', concept of totipotency, Gottlieb Haber-


landt, historical cell division-inducing strategies, historj.' of cell and tissue cultui'e, his-
tory of biology.

.£. Hoxtermann, Miirkische Allee 326, D-J26S9 Berlin, Germany,

„ • t- J ..• • , factors. Sachs proved the cotyledons to be nulnent reser-


O r g a n correlation a n d regeneration in the • r • u l i- r r> /.-.^.,^
^,k«i^ A 1, &•='"'"""" . " ' " ' ^ voirs, confirming the early discovery of Bonnet (1754)
morphology and physiology of germination - the , , ? ,, , , V^
origin of organ ctilture in 19th century botany * ^ ' '•^"°™' "^ cotyledons prevented embryos from
' •> growing.
The first attempts at tissue culture in botanical research Irmisch and S.achs induced many attempts to isolate
originated from morphogenic and physiological studies and culture mature embryos (Gris 1864, van Tieghem
on organ correlation and plant regeneration. The mor- 1873, Marek 1875, Blociszewski 1876) or parts of em-
phologist Irmisch combined research on morphology bryos, especially cotyledons (van Tieghem 1873, Marek
and development and in 1858 he tried to culture parts of 1875, Blociszewski 1876, Zahel 1882) and later hypoco-
ttie embryo (cotyledons) to investigate morphogenic re- tyls (Smith 1907), Even itmnature emhryos developed
lations of the whole to its parts. At ahout the same time, into normal plants (Hannig 1904).
the physiologist Sachs (1859) succeeded in raising iso- The first cultures of vegetative embryos were aimed
lated embryos from mature beans in artificial media. He at studying the morphogenic and physiological behav-
was interested in Ihe physiology of gennination and de- iour of isolated organs and the nutrient and enviionmen-
velopment and studied the morphological and 'chemi- tal requirements for complete regeneration and normal
ca!' (starch-related) differentiation of growing embryos growth, Koch (1887) was able to regenerate whole
and their dependence on histological and environmental plants, even from only a few embryonic cells. In general.
Received 11 January, 1996, revised 3 January, 1997

716 Physiol. Planl. 100,1997


all these culture studies showed the regenerative ability
of embryonic tissues and the reciprocal relationship be-
tween regeneration and differentiation; highly differenti-
ated and specialized tissues couid hardly be raised. The
cultures of diiferentiating embryonic tissues were in a
sense equivalent to experiments on the regeneration of
lower plants (Vochting 1885) and animals (Loeb 1900b)
and did not immediately contribute to the concept of cul-
tures of indefinite growth.
Nevertheless, it was only a small step from the regener-
ation problem to 'true' tissue cultures, Wiesner (1892) di-
rected attention to the limits of divisibility in the vegeta-
tive kingdom with his book on 'The Elementary Stmcture
and the Growth of the Living Substance', His student
Rechinger {1893) took the view that, in principle, the pro-
toplasm as 'the last elementary structure of the living cell'
is the minimal reproductive unit. But in practice, isolated
individual cells would not divide because 'plastic and re-
serve substances' delivered by other celis were lacking,
Gottlieb Haberlandt (Figs 1 and 2), in contrast, saw
some chance of adding these essential substances exoge-
nously. He regarded cells as simple, distinct 'elementary
organisms' or relatively independent 'living units': 'Als
Elementarorganismus „, ist die Zelle eine Lebenseinheit,
ein Individuum fiir sich, das ein vom Gesamtorganismus

Fig, 2, Oil-painted portrait of Haberlandt by the Graz artist


Koko Micoletzky, 1924 (with kind permission of Prof, Dr
Walter Haberiandt, Tubingen),

his zu einem gewissen Grade unabhangiges Eigenleben


fuhrt' (Haberlandt 1925),
Physiological experiments with cells of less organized
plants may have encouraged him in this opinion, Klebs
(1888) had cultured isolated cells of algae to determine
their conditions for living and recommended analogous
investigations with cells of higher plants - advice that
Townsend (1897) took to study the role of the nucleus in
cell wall formation.

Haberlandt's 'Culturversuche mit isolierten


Ptlanzenzellen' - the beginning of cell and tissue
culture
Haberlandt (1902) was the first to attempt the systematic
culture in vitro of somatic cells from higher plants, a
technique for determining the optimum developmental
requirements of different kinds of cells. He suggested
that not only could the properties and potentialities of in-
dividual cells be investigated, but that also some insight
might be gained as to the mutual influences within the
multicellular body: ',,, the results of such culture experi-
ments should give some interesting insight to the proper-
ties and potentialities which the cell as an elementary or-
ganism possesses. Moreover, it would provide infonna-
Fig, 1, Gottlieb Haberlandt (I854-1945), water-eolour, self-por-
trait, ca 1890 (wiith kind permission of Prof, Dr Walter Haber- tion about the inter-relationships and complementary in-
landt, Tubingen), fluences to which cells within a multicellular whole or-

Physiol, Plant, 100,1997 717


ganism are exposed' (Haberlandt 1902; the cited transla- those of cell shape, cell walls, nuclei and chloroplasts.
tions of Haberlandt's paper are taken from Krikorian and Isolated mesophyll cells from Lamittm purpureum and
Berquam 1969), stamen hair cells of Tradescantia virginica started to
Haberiandt used mesophyll cells as well as non-green grow and stayed alive for several weeks. Cell-stretching
cells from different types of trichomes. He preferred and wall-thickening were obsen'cd, but cell division
cells that were loosely orgatiized into tissues and conse- never occurred (Fig, 3), Haberlandt concluded thai fu-
quently easy to isolate mechanically. The cultures were ture attempts at culture would focus on the problem of
initiaUy maintained in hanging-drops on slides and later ceil division: 'It will he the problem of future culture ex-
in small glass dishes containing 'water or different ntitri- periments to discover the conditions under which iso-
ent solutions comprising mineral salts, sucrose, glucose, lated cells undergo division' (Haberlandt 1902),
glycerine, asparagine and peptone. He examined micro- Haberlandt saw certain indications of extemally-in-
scopically-striking morphological changes, particularly duced cell divisions in the experimental findings of Loeb

CIlaTjerlaiidl, {'iiltirrra-Kiirtu' tiul isolierteii ?flari7ra'/unm,

Fig, 3, Plate of Haberlandt


(1902) with fignres of
mesophyll cells (1-5) from
Lamium purpureum, stamen
hair cells (7-10) from
Tradescantia virginica and
glandular hair cells (11-14)
from Pulmonaria mollissima
showing cell growth (1, 2,7),
eell wall thiekening (3—5,
8-10) and vacuolization of
Sitiungsberichte ikais.Akad d 'Wiss , nidth,-naturw,Classe, Bcl,CXI,Abth,l, WOS dying cells (11-14),

718 Physiol, Plant, 100, 1997


(1900a), Nathanson (1900) and Winkler (1901) on artifi- he used cell complexes to get to understand the empiri-
cial parthenogenesis, Loeb and Nathanson had 'pro- cal limits of divisibility: How small could tissue frag-
duced' further deveiopment of unfertilized eggs by ments be and were there special tissue regions particu-
ionic, osmotic or physical means, whereas Winkler had larly qualified for ceil division? 'ich suchte die Frage zu
added extracts from 'male' tissues. Haberlandt pre- beantworten, wie klein die Gewebesttickchen sein kon-
sumed that the active extractable principle was a type of nen, um noch jene Zeilteilungen zu erfahren, die bei
'growth enzyme': 'Probably substances are involved mechanischen Verletzungen der Orgaoe zur Wundkork-
here, 'growth enzymes' which, released from the pollen biiduog fuhren oder die Kallusbildung begieiten. Femer
tube, act as a chemical stimulus !o the growth and divi- wurde die Frage aufgeworfen, ob in den kultivierten
sion of the cells concerned' (Haberlandt 1902), Zellkomplexen ganz bestimmte Gewebearten vertreten
In this case, he distinguished clearly between genera- sein miissen, damit es in ihnen zur Zellteilung kommen
tive and vegetative cell-division stimuli. Referring to konne' (Haberlandt 1913),
Winkler (1901), he suggested that isolated vegetative Haberlandt's new, indirect approach to cell cuiture ied
cells and pollen tubes could be cultured together: 'Still, to a series of communications on the physiology of celi
it would be worthwile to culture together in hanging divi.sion. Already in 1913 he experimentally proved the
drops vegetative cells and pollen tubes; perhaps the lat- existence of ceil division factors in plants. He envi-
ter would induce the former to divide' (Haberlandt sioned cell division and periderm formation as being
1902), By suggesting the use of what is essentially a regulated by two hormones: the so-called 'lepto-hor-
combined culture, Haberlandt anticipated what later mones' that were associated with the vascuiar tissue
came to be known as the 'nurse culture technique'. Sin- (1913), and the 'wound' or 'necro-hormones' that were
gle cells can be grown on previously cultured explants, secreted by injured or dead cells, respectively (1921a,b).
which release growth stimulants. As a major result, Haberlandt (1922) formulated the
Expecting that growing tissues or embryonic media concept of a cell division hormone embracing various
produce growth-promoting substances, he moreover developmental phenomena such as wound heaiing, fer-
proposed the application of extracts from vegetative api- tilization, parthenogenesis or adventitious embryo for-
ces or, alternatively, the imitation of nearly natural con- mation. His students Lamprecht (1918) and Reiche
ditions by using embryo sac fluids: 'Not only pollen (1924) confirmed this idea (Hoxtermann 1996b), Thus,
tubes could be utilized to induce division in vegetative Haberlandt established the first hormone theory' in bot-
cells. One could also add to the nutrient solutions used any (Hoxtermann 1994a,b) which eventually culminated
an extract from vegetative apices, or else culture the with the discovery of kinetin (Miller et al. 1955) and
cells from such apices. One might also consider utiliza- other cytokinios (Skoog et al, 1965),
tion of embryo sac fluids' (Haberlandt 1902).
Thus, with a few sentences Haberlandt predicted the
First cultures of meristematic root tips and the
future direction of cell and tissue culture. Nurse cultures
estahlishment of 'true' organ culture
and cultures of root and shoot apices are now a reality,
and embryo sac fluids turned out to be rich sources, of New experiments with photosynthetic tissue cultures
cell division-inducing hormones. He reduced the culture (e.g. Bobilioff-Preisser 1917) confirmed Haberlandt's
problem to that of cell multiplication and drew attention observations: more or less growth, but no ceil division!
to hypothetical 'growth enzymes' or 'hormones' that The situation changed by luming from parenchymatous
were as yet unknown. to meristematic tissues. Robbins and Kotte indepen-
The theoretically most important aspect of Haber- dently of each other succeeded in culturing tips of Pisum
landt's paper appears to be his premature and daring and Zea roots in different synthetic media. Robbins
general conclusion that all living cells, even somatic (1922a) was especially interested in nutritional and cor-
protoplasts, were what has since come to be called 'toti- relative questions, whereas Kotte (1922a,b) pursued
potent': 'Without permitting myself to pose further morphogenic problems,
questions, I believe, in conclusion, that I am not making Robbins wished to study the complete and uninflu-
too bold a prediction if I point to the possibility that, in enced nutrient requirements of roots and shoots so as to
this way, one could successfully cultivate artificial em- characterize their interactions. He liked '.., to define the
bryos from vegetative cells' (Haberlandt 1902), classes of materials required by a plant shoot or root for
Haberlandt had performed his culture experiments as continued growth. ... In order to eliminate the influence
early as 1898 with the original intention to pursue them of the shoot aod its products on the root, or of the root on
on a large scale. But his investigations of sensory physi- the shoot, it was considered necessary to grow ... tips
ology (Haberlandt 1904, 1908) prevented this from be- isoiated ..., in artificial media under sterile conditions'
ing carried out and so he briefly communicated his ear- (Robbins i922a). He attached particular importance to
lier results in 1902. absolute sterility - a necessity ',,, in the investigation of
Only in 1911, after his appointment at Berlin, did he problems involving the direct use by higher plants of or-
return to the problem of cell culture. Changing his strat- ganic or inorganic substances which may be altered by
egy, he no longer worked with individual celis. Rather, bacteria] action' (Robbins 1922a).

Physiol. Plant. IOO, 1997 719


In contrast, Haberlandt had not thought it possible or morphogenic influences that were believed to be analo-
necessary to achieve completely aseptic conditions: 'Vari- gous to internal developmental factors,
ous precautions were taken, of course, to Iteep the cultures Robbins and Kotte established the important fact that
as nearly bacteria and fungus-free as possible, though in an excised root meristem, if it grows, continues to differ-
this regard, complete sterilization turned out to be scarcely entiate into the usual tissues and to develop as a nonnal
feasible and really unnecessary' (Haberlandt 1902). root. Thus, they are credited with the firsl successful or-
However, sterile culture was demanded by plant pa- gan culture experiments.
thologists to study the relationships between a pathogen Both introduced colloidal agar media as being supe-
and its host. Prompted by their contact with Benjamin rior to liquid cultures. They also confirmed the intention
Duggar, Knudson (1919) and Robbins (1922a) exploited of Haberlandt that the addition of crude extracts may im-
aseptic culture techniques in their work on root caps and prove the nutrient medium, Robbins used yeast autolyz-
tips, respectively. ate for this purpose, whereas Kotte obtained beneficial
Robbins performed his investigations in 1917, at effects with meat extract.
which time he was unaware of Haberlandt's experi- In the cultures of Robbins and Kotte, active growth
ments. He designed his studies to examine the hypothe- increments did not occur, as only comparatively shori
sis of Loeb (1917) that the development of roots in leaf culture periods were employed. In the following years
notches of Bryophyllum was induced by a hormone of an extensive series of investigations into satisfactory nu-
the leaf: 'It seemed ,,, that Loeb's hypothesis could be trient conditions was undertaken to continue the indefi-
tested by comparing growth of excised root tips in a so- nite growth of excised organ tips (Fiedler 1938/39,
lution of mineral salts witb their growth in one of mitti- Street 1959), Great strides were made by systematic
eral salts and sugar. Growth in the latter medium, if it oc- tests of (1) carbohydrates (glucose, Robbins 1922a,
curred, would demonstrate that sugar was the 'hormone' Kotte 1922a; sucrose. White 1934), (2) amino acids (as-
furnished by the leaf and necessary for the growth of paragine, glycine, peptone, Robbins 1922b, Kotte
roots in the leaf notches' (Robbins 1957), 1922a; complementary mixtures, Bonner and Addicott
Kotte (Fig. 4) was a student and assistant of Haber- 1937, White 1937a), (3) hormones (concentration-de-
landt and wished to test the general growth potential of pendent effects of auxins, Fiedler 1936, Bonner and Ad-
meristematic tissues by means of tissue culture. Only dicott 1937), (4) tissue extracts (from seedlings and em-
permanent tissues had been investigated previously: bryos, Robbins and Maneval 1923), and (5) yeast ex-
'Bisher wurden nur Dauergewebe auf diese Weise unter- tracts (inaugurated by Robbins 1922b and then effec-
sucht, wabrend isolierte medstematische Gewebe an- tively used by White 1934 and Bonner and Addicott
scheinend noch nicht in Kultur genommen worden sind' 1937). The active principles of tissue extracts were as-
(Kotte 1922b). He considered tissue cultures to be a 'de- sumed to be hormonal growth substances, whereas the
velopmental-analytical method' for studying external remarkable growth-promoting activity of yeast extracts
was attributed to the existence of essential trace ele-
ments (Robbins and Wbite 1936, Fiedler 1936), vitamins
(especially vitamin Bj or thiamine, Bonner 1937, White
1937b) and amino acids (Bonner and Addicott 1937),
By using those exogenous ingredients White (1934)
was able to announce tiie 'Potentially unlimited growth of
excised tomato root tips in a liquid medium'. He worked in
a department of pathology and preferred to study physio-
logical problems with a pathological background. Tomato,
for instance, was chosen as a test organism for later studies
of certain diseases. Most of the plant tissue cultures re-
ported in literature were pathological cultures (as were the
experiments of Haberlandt), showing only survival, often
for months, but not true growth. White outlined, for the
first time, the nutritional requirements for real, permanent
growth. He distinguished between survival with minimal
metabolic rates and a medium acting essentially as an inert
substrate and growth with high metabolic activity at the
expense of the culture medium. White convincingly
showed that his nutrient supply was adequate for all
Fig. 4, 'Walter Kotte (1893-1970), Haberlandt's assistant from growth requirements. Extending the earlier work of Rob-
1920 to 1922 and one of the co-founders of plant organ culture, bins (1922b), he demonstrated in particular that oiinute
in 1956 (with kind permission of the Archives of the German amounts of yeast were beneficial and even necessary.
Academy of Scientists Leopoldina in Halle/Saale, file 'MM
4929'). Kotte later changed from developmental physiology to In the 193O's mucb work was done to identify the un-
plant pathology. known 'active principles' of yeast and lo substitute them

720 Physiol. Planl. 100, I » 7


with known chemical substances, Bonner and Addicott small, compact cell aggregates in the presence of a con-
(1937) replaced yeast extract with a complementary centrated leaf extract, 'Dieser theoretisch und praktisch
mixture of vitamin Bi and diverse amino acids and re- wichtige, neuartige Befund laBt vielleicht sogar auf die
ported sustained growth of excised pea roots in the first Totalregeneration der ganzen Bliitenpflanze aus einer
completely known artificial medium, Bonner and Addi- Zelle hoffen' (Schmucker 1929), Schmucker was prima-
cott, working in a laboratory' of biological science, were rily interested in ne'w methods and presented an epider-
interested in 'Plant tissue cultures from a hormone point mal model for transpiration studies and a simple, me-
of view' (Bonner 1936), The progress in auxin research chanical isolation method for obtaining individual cells.
formed the basis of their culture experiments: 'Because Unfortunately, he did not pursue his culture experiments
of the work on auxin we are now beginning to realize the and remained unnoticed.
great part special chetnical substances or 'hormones' Since tissue culture attempts with different plant ma-
play in piant growth and development, and it seems terial failed, methodological aspects were increasingly
probable that such substances must be present in any considered to be the cause of this persistent failure. Ev-
medium in which plant tissue is to be successfully culti- ery effort was made to improve isolation, sterilisation
vated' (Bonner 1936), 'It was 'with the hope of isolating and culture. Special attention was paid to the nutrient
and identifying still other substances concemed with media, to their physical state (e,g, to nature-like
growth and differentiation that the present work was un- semi-solid agar gels, already used by Hannig 1904, in
dertaken' (Bonner and Addicott 1937), contrast to liquid solutions; and to optimum hydrogen
After the estabhshment of a suitable technique for ion concentrations, Pearsall and Priestley 1923), as well
continuous root growth, Bonner's and Addicott's cul- as to the chemical and biological components of the me-
tures of excised organs became a valuable, multipurpose dia. The interest became progressively focussed on ac-
standard method for physiological and biochemical in- tive factors of tissue extracts.
vestigations. In cultures of root tips the meristematic tis- In 1927 Wehnelt, one of the last student,s of Haber-
sue continues to differentiate in a comparatively normal landt in 1923/24, reported a sensitive technique for test-
fashion. Tissue culture without organ generation was ac- ing cell division activity. Dealing with the problem of
complished only by Schmucker (1929) with palisade wound healing, he found the homogeneous, vascu-
cells, and by Gautheret (1937) with cambial cells, but lar-free pericarp tissue inside the immature hulls of
their results were difficult to confirm. young beans to be suitable. Drops of tissue extracts in-
duced a callus-like proliferation of only parenchyma
cells, 'In jedem Falle handelte es sich bei den durch die
The long, winding route of plant tissue ctilture and wirksamen Stoffe hervorgerufenen Neubildungen um
the problem of cell division rein parenchymatische Gewebe ,,,' (Wehnelt 1927),
After Haberlandt (1902) had made a research subject of Extending the investigations of Wehnelt (1927), Bon-
cell and tissue culture, but failed in his attempts with leaf ner (1936) cultured pericarp tissue from bean pods. He
cells and trichomes, which were easy to isolate mechani- succeeded using an alcoholic hull extract. The cells both
cally but highly specialized, his successors tried to find enlarged and divided and continued to grow as a callus
more suitable objects, Winkler (1902) and Bobilioff-Prei- without any differentiation! Though the growth was lim-
sser (1917) used leaf parenchyma from other species and ited after a time, Bonner could verify a reproducible tis-
Haberlandt's visiting scholar Thielmann (1925) exam- sue culture for the first time. He was convinced that the
ined epidermal cells, Stomatal cells proved to be particu- growth-promoting substances might be derived from ex-
larly viable and stayed alive up to four months. Referring ogenous sources, 'The objectives of the present work
to an obser\'ation by Leitgeb (1888), culture experiments have therefore been the separation and purification of
with guard cells had already been recommended by Ha- special substances or hormones which control growth
berlandt: 'In any case, the stomatal cells, because of their and development of plant tissues and organs, and the
great viability, suggest themselves as very suitable for petfection with the aid of such substances of true tissue
this kind of culture experiment' (Haberlandt 1902), cultures' (Bonner 1936),
Apart from leaf tissues, a variety of other plant tissues Further concentration on methodology brought the
was tested, e,g, root-cap cells (Knudson 1919), cells breakthrough. Early in 1939, three workers. White in the
from cryptogams, ovaries and fruit pericarp (Borger USA, and Gautheret and Nobecourt in France, published
1926, Pfeiffer 1931), perianth cells of flowers (Kunkel independently of each other and within a period of sev-
1927), marrow cells of stems (Ulehla 1928) or cells from eral weeks the results of continuously growing tissues,
the cambium and its derivative tissues (Bailey 1930), As with the consequence that two distinct research hnes
mentioned, changing from parenchymatous tissues to arose: an American and a French school of plant tissue
root tips opened the new field of organ culture. culture with different objectives, materials and tech-
The first, and for a time only, note of a successful cell niques (White 1946, Stapp 1947, Kandler 1950),
culture was that by Schmucker (1929) who announced in White favoured problems dealing with cancer re-
passing that isolated mesophyll cells of a poppy species search. After Stapp (1927) had found that bacteria-in-
{Bocconia cordata) would divide and develop into duced tumours of plants acted much like malignant can-

Physiot, Plant, tOO, 1997 721


cer tissues. White connected the general tumour problem
with the topic of tissue culture and produced callus cul-
tures from both stem tissues of tumour-forming tobacco
hybrids containing procambium (White 1939) and
crown-gall tumours of sunflower (White and Braun
1942), The tumorous cultures could be transplanted and
upon implantation produce new, secondary tumours in
healthy hosts. White (1939) was interested in analysing
factors involved in controlling differentiation of Nicoti-
ana tumour cultures — an approach that especially Skoog
later continued with, Skoog stated: 'It is possible to con-
trol undifferentiated growth and organ formation in to-
bacco callus cultures ,,, by manipulation of external fac-
tors ,,,' (Skoog 1944),
In the USA, cultttres of undifferentiated masses con-
sequently became restricted to neoplastic tissues derived
from several types of aseptic tumours. In France, on the
other hand, cultured tissues from normal, non-tumorous
plant materials were studied with special regard to mor- Fig, 5, The cell division-promoting effect of coconut milk on the
phogenesis. After the first, very frail cultures of growth of carrot caltures, as Haberlandt (1902) had predicted:
slowly-growing cambial tissues from woody plants (1) Pieces of phloem tisstie after 21 days on a basal nutrient agar
(Gautheret 1937) Gautheret (1939) in Paris and Nobe- medium, (A-D) with indoleacetic acid at various concentia-
tions, (E—H) with different quantities of coconut milk (Caplin
court (1939) in Grenoble succeeded in cuituring cambial and Steward 1948),
root tissues of carrot. Their work dealt with the effects of
nutritional and morphogenic factors, particularly of the
indoleacetic and naphthaleneacetic acids (Gautheret) as the liquid endosperm of the coconut, Letham had also
well as indoleacetic acid and vitamin Bi (Nobecourt), on isolated the first native cytokinin, zeatin from immature
growth, development and differentiation in vitro (root maize grains, in 1963, half-a-century after Haberlandt
and shoot initiation, formation of vascular tissues, bud- (1913) had experimentally demonstrated the existence of
ding, etc). Special emphasis was paid to the problem of hormonal cell division substances in plants.
tissue polarity (in the movement of heteroauxin) and to The necessity of essential vegetative supplements
the morphological responses resulting from this polar- (yeast extract or coconut milk) to cultured tissues was
ized movement (Gautheret 1942/43, Nobecourt 1943), surmounted when Reinert (1958) introduced a fully-syn-
Gautheret (1942) expressly discussed the bearing of the
thetic culture medium. The formation of roots and
various findings on the hormone theory of Haberlandt,
shoots in callus tissue from carrot became controllable
The first successes of White, Gautheret and Nobe- by classic one-factor-analysis, pointing the way to artifi-
court were not achieved with cultures of individual cells, cial embryogenesis of single somatic cells (Reineri
but from relatively large tissue explants containing cam- 1963, Steward etal, 1966),
bium. Culture of small tissue pieces, sufficiently re-
moved from the cambium, proved to be much more dif-
ficult and was only possible in 1948 when coconut milk Animal tissue culture - a rapidly-attained goal
was used as supplement (Fig, 5), Coconut milk then be- despite a delayed start
came frequently used in basal media following the suc- Fiedler (1938/39), probably somewhat resignedly, em-
cess of van Overbeek and co-workers (1941) in growing phasized the wide gap between plant and animal tissue
immature Datura embryos by including coconut milk in culture: 'Es ist an und fiir sich sehr merkwUrdig, dafi
their culture medium. Subsequently, the coconut's liquid man das Pflanzengewebe, das im Vergleich zum
endosperm was shown to promote cell division in other tierischen viel einfacher gebaut ist, isoliert nicht zum
tissues, as Haberlandt (1902) had predicted. Coconut weiteren Wachstum bringen kann, wahrend den Tier-
milk recommended itself since relatively large volumes physiologen, die ,,, mit einem viel komplizierieren Aus-
are readily available in a fluid state. It is tempting to gangsmaterial rechnen muBten, schon nach kurzer Zeit
speculate ',,, that perhaps Haberlandt himself might eine (erfolgreiche, E, H,) Erfahrung zur Verftigung stand
have conceived coconut as being a source of readily ,,,' (Fiedler 1938/39),
available 'embryo sac fluids', had coconuts been gener- Animal tissue culture has its roots, just as has plant
ally available in Berlin' (Krikorian and Berquam 1969), tissue culture, in the physiology, morphology and em-
Since the 195O's, a great deal of work was done on the bryology of the 19th century. Already the concept of the
nature of the coconut milk growth factors, Letham internal and external milieu of organisms, organs, tissues
(1974) identified cytokinins, i,e, cell-division inducing and cells by Bernard (1878) had given an impetus to
plant hormones as Haberlandt (1902) had expected, in study tissues and cells under extracorporal conditions.

722 Physiol, Planl, 100, 1997


Much of the pioneer work was done using embryonic as to whether cultures showed actual hving activity or
tissues on account of their more rapid growth. The early artificial survival and warned against the too facile ex-
attempts at best achieved cell survival without actual trapolation from in vitro findings to in vivo events.
growth, Ljunggren (1897/98) then successfully cultured The development and scope of animal tissue culture
human tissues. He grew skin epithelium and showed that were significantly infiuenced by two factors: first, the
even under conditions in vitro a comparatively normal increased progress in biochemisti'y and second, the ob-
histology could be preserved over a certain time. servation by Fleming (1929) of the antibiotic action of
The anatomist Harrison (1907), for the first time, used penicillin. The more or less empirical and quahtative ap-
tissue culture to solve a selected developmental prob- proach to tissue culture merged imperceptibly into more
lem, i,e, the origin of the axon, which had been in dis- planned and quantitative methods. The general applica-
pute for some years, Harrison intended ',,, to obtain a tion of tissue culture techniques came with the introduc-
method by which the end of a growing nerve could be tion of relatively non-toxic antibiotics in the 194O's,
brought under direct observation while alive' (Harrison which could be added to culture media and thus allowed
1907) and could demonstrate impressively that by cul- those not specially trained in bacteriology or surgery to
turing pieces of frog embryos in a drop of lymph the use these methods. From then onwards cultured tissues
axon was an outfiow of the central neurone cell, contributed fundamentally to solving problems posed by
Harrison presented the first efficient tissue culture medical and veterinary science, and cell and microbiol-
technique in the history of biology. He continued former ogy (Wylie 1967),
embryologica! regeneration and explantation studies by In Germany, Rhoda Erdmann generally promoted
Roux (1895), Driesch (1897) and J, Loeb (1900b), but both animal and plant tissue culture when she founded a
also implantation experiments with tissue 'cultures' in Department of Experimental Cell Biology in Berlin
warm-blooded bodies by L, Loeb (1897) and others (1919) and edited the 'Archiv fur experimentelle Zell-
(Bucher 1940), Although a zoologist, Harrison was fa- forschung, besonders Gewebezuchtung' from 1925 on-
miliar with the early work on plant cells. Without envy, wards. She brought medical practitioners, zoologists,
he admitted Haberlandt's priority in systematic attempts botanists and bacteriologists, anatomists, physiologists
at tis:sue culture under extraorganisniic conditions: and pathologists together (Hoppe 1989), In the first vol-
'About the time that Haberlandt's first paper appeared ume of the 'Archives', Haberiandt's students Lamprecht
Loeb published his experiments with pieces of epithe- (1925) and Thielmann (1925) reported on their results of
lium from the Guinea pig, imbedded in small blocks of plant tissue culture.
clotted blood or agar which were placed for incubation Interdisciplinary relations and interactions between
in the body of another animal, ,,, The technique was en- animal and plant tissue culture research were typical for
tirely different from Haberlandt's and had nothing in that time. The zoologist and anatomist Roux, for exam-
common with that of modem tissue culture, although the ple, edited a 'Terminology of the Mechanics of Develop-
underlying puipose of the experiment was essentially the ment' (1912) of both animals and plants, in close con-
same, Loeb in an earlier paper ,,, mentions having made nection with the botanists Correns and Klister, As stated
experiments in which the agar blocks containing pieces earlier, even Haberlandt (1902) had relied on the zoolog-
of living tissue were incubated outside the organism. ical findings of Loeb and Nathanson and the root culture
The results were not stated' (Harrison 1928), experiments of Robbins (1922) had their immediate ori-
Tissue culture in experimental medicine and zoology gin in the ideas of Loeb (Robbins 1957),
was tackled later than that in botany but was successful It is an interesting historical fact that the first tissue
much sooner. The objective of permanently-growing, culture attempts in botanj' as well as in zoology arose
non-differentiating individual cells was quickly reached. from experimental anatomy, Haberlandt and Harrison,
The morphogenesis of peripheral nerve fibers was al- both anatomists, led the descriptive and comparative
most completely a subject of descriptive embryology, anatomy and ontogenesis research of the 19th century to
but opened a wide field of causal-analytical investiga- the progressive causal-analytical physiology of develop-
tions into problems of the physiology of development. ment of the present one.
Within only a few years an efficient technique was
elaborated, especially by Carrel (1914), Frog lymph was
Cellular totipotency - the daring vision of
replaced by chicken plasma and embryonic and various
Haberlandt
organ extracts were found to be strongly growth-stimu-
lating. Carrel also surmounted the hazards of infections. Already as a young postgraduate, Haberlandt had con-
As an experimental surgeon he knew the methods of an- ceived his concept of a physiological interpretation of
tisepsis and asepsis. Using chicken plasma he kept plant anatomy, which Schwendener (1874) had sug-
chicken fibroblasts alive for over 30 years. But in spite gested. In 1884 his 'Physiologische Pflanzenanatomie'
of its furious start, animal tissue culture was only slowly appeared, Haberlandt's approach was to reach conclu-
adopted. The rigid demands made by sterility and the sions about the physiological functions of anatomical
new paradigm of studying biological processes outside structures oti the basis of purely microscopical observa-
the organism caused trouble. Opponents were sceptical tions (Hoxtermann 1996a), Most of his deductions stim-

PhysioL Plant, 100, 1997 723


ulated physiological research and were confirmed by et a], 1958) were applied. But the most significant result
later experiments, as were also his attempts at tissue cul- was his far-reaching postulation of what has come to be
ture, called 'totipotency',
Haberlandt was both a thorough obser\'er and a splen- Haberlandt believed it possible that artificial embryos
did and daring interpreter, 'He had eminendy what the could be successfully cultured from somatic cells. The
Germans call a philosophical mind, never being satisfied word 'totipotence' seems to have been used first by
with the establishment of mere facts, but always at- Morgan (1901), Various examples of vegetative propa-
tempting a theoretical interpretation' (Noe 1934), In par- gation in the botanical and horticultural literature legiti-
ticular, two theoretical positions formed the basis of his mized the conceptual adoption of totipotency from ani-
tissue culture experiments: first, he regarded single cells mal embryology and its transfer to the botanical physiol-
as 'elementary organisms' (a term that had been intro- ogy of development. Van Tieghem and Douliot (1888),
duced by Briicke 1862), and second, he acknowledged for instance, had investigated root-forming tissues. Al-
the existence of 'morphogenic substances' and chemical though roots appeared to arise most frequently from the
correlations between these elementary units. pericycle, root initials were observed to be formed in al-
In his 'Physiological Plant Anatomy' he emphasized most any living tissue. Moreover, Winkler (1903) dis-
that cells were units not only in the morphological sense: covered the impressive phenomenon that adventitious
'If the term 'organ' be employed in general to denote the buds formed on leaves may originate either from several
instrument wherewith a definite physiological function cells together or even from a single epidermal cell.
is performed, then the cell must be regarded as an ele- However, the long-lasting failure of many tissue cul-
mentary physiological instrument or 'elementary organ'! ture attempts nourished doubts about the developmental
Every cell, namely, performs a definite physiological potential of somatic cells, Kuster (1928) questioned
service for the whole term of its life or at any rate at whether somatic tissue culture was perhaps principally
some period of its existence while the sum total of the impossible because the types of cells used were unable to
physiological functions of the various cells constitutes develop, Miehe (1928) also took the view that differenti-
the vital activity of the entire plant, ,„ The majority of ated cells from permanent tissue could not regain a juve-
cells represent not only elementary organs, but also ele- nile status: ',„ die sogenannten Dauerzellen (sind) grund-
mentary organisms, in other words, a cell, as a rule, does satzlich nicht imstande, in den entwicklungsfahigen
not merely work in the service of a higher living entity, Zustand zuriickzukehren' (Miehe 1928), He reproached
namely the entire plant, but also behaves as a living en- botanists like Haberlandt for misunderstanding the plant
tity, though indeed as one of a lower order of magnitude' body as a kind of colony of relatively independent cellu-
(Haberlandt 1914), Haberlandt suggested that the recip- lar sub-individuals. The somatic cells would have 'some-
rocal relationships between the cellular 'elementary or- thing', a special type of protoplasm, the so-called 'ar-
ganisms' might well be studied through observations of chiplasm', missing. The archiplasm-hypothesis of Miehe
the results obtained by severance of these correlations, to a ceriain extent followed the germ plasm theory of the
that is, by the culture of isolated cells and tissues in vitro zoologist Weismann (1885), Miehe rejected the repro-
- an idea, first clearly formulated in 1902, ductive equivalence of the subindividuals of the so-called
Haberlandt was not successful. Besides a review by 'cell state', i,e, the principle of totipotency.
Winkler (1902), httle immediate notice was taken. The But some experimental findings spoke against this hy-
now classic paper was not even mentioned in an appreci- pothesis, Haberlandt's student Reiche (1924), for in-
ation on the occasion of Haberlandt's 80th birthday (Noe stance, induced new tissue formation of somatic cells by
1934), The situation changed around 1940 when plant injected tissue juices. Moreover, the successes with ani-
tissue culture came into being. In the 196O's the pioneer mal tissue cuitttres encouraged botanists to disapprove
work of Haberlandt became so popular and so often theoretical discussions on the impossibility of cultures
quoted that in 1969,67 years after its appearance, Kriko- from fully-differentiated tissues and to search for better
rian and Berquam presented an entire English translation methods.
of the original paper, 'The paper contains theoretical The term 'totipotent' was popularized among botanists
considerations which hindsight now shows to have been especially by Sinnott (1950, 1960), In his book 'Cell and
particularly accurate' (Krikorian and Berquam 1969), Psyche, The Biology of Purpose' he wrote ',,, the general
Although success first came with animal tissues, the conclusion, with all its far-reaching implications, seems
botanist Haberlandt clearly set forth the purposes and po- justified that every cell, fundamentally and under proper
tential of cell cultures and foresaw their usefulness as an conditions is totipotent, or capable of developing by re-
elegant means of studying morphological and physiolog- generation into a whole organism' (Sinnot 1950), 'The
ical problems. His paper ushered in a new era of research, fate of a cell is a function of Its position, ,,, Single cells,
Haberlandt anticipated both future methodological under suitable conditions of isolation and stimulation,
and theoretical developments. Experimental ideas were will sometimes develop into whole plants. All paris of
realized when extracts irom vegetative apices, embryo the plant tend thus to be totipotent. Why these potentiali-
sac fluids that promoted growth and cell division (van ties are not realized when the part is a member of an or-
Overbeek et al, 1941), or nurse culture techniques (Muir ganic whole is a problem' (Sitmot 1960),

724 Physiol, Plant, 100, 1997


Tab, 1, Review: Plant organ, tissue and cell cultures.

The culture of isolated embryos or embryonic pans


1858 Thilo Irmisch (181^1879), Soadershausen:
Formulation of the organ culture approach to embryogenesis research
1859 Julius Sachs (1832-1897), Prague:
First culture of isolated embryos from mature seed (beans)
1887 Eudwig Koch (1850-1938), Heidelberg:
Regeneration of whole plants from only few embryonic cells of seedlings (Orobanche)
1904 Emil Hannig (1872-1955), Strasbourg:
First culture of immature embryos
1907 Eouie H, Smith (1872-? ), Halle/Saale:
First culture of excised hypocotyls

The beginning of plant cell and tissue culture


1893 Carl Rechingei (1867-1952), Vienna:
Extensive tissue culture studies of the limits of divisibility
1902 Gottlieb Haberlandt (1854-1945), Graz:
First systematic culture attempts with individual somatic cells and general formulation of the culture programme
i9I3 G, Haberlandt, Berlin:
First experimental evidence of cell division-stimulating substances, establishment of a lepto-hormone theory of cell division
1921 G, Haberlandt, Berlin:
Establishment of a wound-hormone theorj' of cell division

The culture of isolated root tips


1922 William J, Robbins (1890-1978), Columbia/Missouri:
First successful culture of a tvpically meristematic tissue to study nuuitional problems
1922 Walter Kolte (1893-1970), B'erJin:
Successful culture of meristematic tissue independently of Robbins to study moiphogenic problems
1934 Philip R, White (1901-1968), Princeton/New Jersey:
First cultures of excised root tips with potentially unlimited growth excluding any pathological survival effects
1937 James F, Bonner (1910) and Fredrick T, Addicott (1912), Pasadena/California:
Sustained growth of excised root tips in the first completeiy known medium

The culture of plant cells and tissues


1927 Bruno Wehnelt (1902), Erlangen:
Report on a sensitive technique for testing cell division activitj'
1929 Theodor Schmucker (1894-1970), Gotnngen:
First note of a successful culture of multiplying single somatic cells
1936 J, F, Bonner, Pasadena/Califomia:
FoUowing Wehnelt 1927, first verification of a reproducible cultui'ed tissue with dividing and enlarging cells and without any
differentiation
1939 R R, White, Princeton/New Jersey:
Indefinitely growing callus cultures from stem tissues of tumour-forming tobacco hybrids (to study pathological phenomena)
1939 Roger J, Gautheret (1910), Paris:
Permanently growing cultares of cambial tissaes from woody plants and of root tissues from carrots (to study morphogenesis,
polarization and intercellular correlation)
1939 Pierre Nobecoar! (1895-?), Gienoble:
Indefinitely growing cultures of root explants from carrots (to study nutritional effects on growth and development)
1942 P, R, White and A, C, Braun, Princeton/New Jersey:
Transplantation experiments with callus cultares from crown-gall tumours of sunflowers producing new, secondary' tumours in
healthy hosts
1948 Samuel M, Caplin (1917) and Frederick C, Steward (1904), Ne'B' York:
Convincing evidence for an active principle in coconnt milk promoting growth and development
1958 Jakob Reinert (1912), Tubingen:
Fully-synthetic cell and tissue culture without any essential vegetative supplements

Reinert (1963) and Steward and co-workers (1966) and palisade cells, capable of synthetis, should be able to
were the first to confiim Haberlandt's prediction that complete the supply of nutrients. The simple nutritional
embryos can arise from single cells in culture. The old medium containing mineral salts and a few organic com-
opinion that genie material is successively altered or lost pounds could not be expected to supply all essential sub-
during the differentiation process had to be discarded, stances. Cell walls, in addition, would restrict and limit
But why was Haberlandt unsuccessful in growing iso- nutrient uptake. Besides, Krikorian and Berquam (1969)
lated plant cells? White (1963) accounted for Haber- pointed out that Haberiandt's technique of excision
landt's limited success in the use of fully-mature and would, often, produce protoplasts that would be ren-
highly-differentiated cells whose meristematic activity dered into a state of shock,
was minimal. These specialized cells were selected for It was optimistic of Haberlandt to think that single,
ease of isolation and for nutritional reasons, Mesophyll highly-differentiated cells could be cultured by simple

Physiol, Plant, 100, t997 '^25


Fig, 6, 'Auttimnal tints in
October', one of the few
water-coioui's of Haberlandt
that were saved, painted In
1940 in Barenfels/Saxony
(with kind permission of Prof,
Dr Claus Schnarrenberger,
Berlin),

mechanical and nutritional means. With maximum faith References


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Edited by C, H, Bomman

728 Physiol, Plant, 100,1997